This research involves the biological approach to the treatment of hearing loss. It is designed to investigate the mechanisms involved in mammalian hair cell regeneration. The proposed experiments build upon the observation that global expression of the Atoh1 gene in cochlear cells is sufficient for hair cell genesis. This proposal will test the hypothesis that specific populations of cochlear supporting cells are able to differentiate into hair cells. To accomplish this, Atoh1 will be expressed 1) in a dose-dependent manner, and 2) specifically in the cochlear supporting cells that reside directly beneath the hair cells. The basic experimental protocol will be as follows. Cultured murine organs of Corti will be electroporated with an inducible form of Atoh1. Atoh1 expression will be limited to the supporting cells directly beneath the hair cells because it will be placed under the regulatory control of the GFAP promoter (selectively expressed in the inner phalangeal, inner border, pillar and Dieters'cells) and GLAST promoter (inner border and inner phalangeal cell). Next, hair cells will be killed and then Atoh1 will be upregulated in these specific supporting cells. The ability for these specific supporting cell types to develop into hair cells will be observed using time lapse video microscopy, electron microscopy, and immunohistochemical expression of hair cell specific markers such as myosin 7a. Relevance: The long-term goal of this research is to treat hearing loss with more effective therapies than provided by hearing aids or cochlear implants. Specifically, this research is designed to investigate the basic mechanisms by which the auditory sensory cells, or hair cells, can regenerate in mammals. The overall hypothesis is that replacement of lost hair cells will result in a superior therapy for the treatment of hearing loss.